General overview of MLL-AF9 experiments .
We employed a mouse model of MLL-AF9 leukemia to investigate the role of Prdm16 isoforms in leukemia. As described in Section 2.2.2, this was the first mouse model of MLL-rearranged leukemia to be developed and is often used because of its relative genetic simplicity and reliable recapitulation of AML364.
We generated MLL-AF9 cells by transduction of a retroviral vector expressing MLL-AF9 fusion protein and a human NGFR reporter (Figure 5-1A). Immortalized hNGFR+ MLL-AF9 cells were expanded in vitro
supplemented with SCF, IL-6 and IL-3. After 3-4 days of expansion, 2x104 hNGFR+ cells (Figure 5-1B) were
transplanted into irradiated recipient mice together with 2x105 supporting WT bone marrow cells
(schematic in Figure 5-1C). Development of myeloid leukemia was confirmed by staining bone marrow for hNGFR+Mac1+Gr1+ cells (example in Figure 5-1D) and by hematoxylin/eosin staining of peripheral blood
(Figure 5-1E). In vitro colony assays were performed with 1000 MLL-AF9 cells in methylcellulose media and assayed after 7 days. All biological replicates were derived from independent MLL-AF9 cell lines originating from different retroviral transductions.
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Figure 5-1: Overview of MLL-AF9 retroviral transduction and mouse transplants. (A) Map of retroviral
MLL-AF9 vector under an MSCV promoter and co-expressing human NGFR. (B) Representative flow cytometry plot showing hNGFR expression after transformation of HSCs with MSCV-MLL-AF9-hNGFR retroviral vector. (C) Experimental design of MLL-AF9 leukemia studies. (D) Representative flow cytometry plot showing hNGFR and myeloid (Mac1, Gr-1) marker expression in peripheral blood of moribund mice at the endpoint of survival experiments. (E) Representative hematoxylin and eosin (H&E) staining of purified hNGFR+ cells from moribund mice transplanted with either Prdm16fl/fl.Vav-Cre (Cre+/-) or WT littermate (Cre-/-) MLL-AF9 cells.
To determine the effect of Prdm16 deletion on leukemogenesis, we retrovirally transduced bone marrow HSCs (Lin-cKit+Sca1+Flt3-) from Prdm16fl/fl.Vav-Cre+ mice and WT littermates with MLL-AF9. Leukemic latency was significantly extended in mice transplanted with Prdm16fl/fl.Vav-Cre+ compared to WT MLL-AF9 cells (Figure 5-2A). Despite accelerating development of leukemia in vivo, colony forming assays showed that Prdm16 did not affect proliferation in vitro, with no difference between Prdm16fl/fl.Vav-Cre+ or WT MLL-AF9 cells (Figure 5-2B). Leukemic cell-of-origin can have a significant impact on resulting leukemia. To investigate the effect of Prdm16 deletion on MLL-AF9 cells generated from other cells-of-origin, we transduced Lin-Sca1-Kit+ progenitors (a population containing GMPs, CMPs, and MEPs)
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with MLL-AF9. Mice transplanted with these Prdm16fl/fl.Vav-Cre+ cells also had a longer leukemic latency compared to WT, although both cohorts had increased latency compared to mice receiving HSC-derived MLL-AF9 cells, a finding that is consistent with published data (Figure 5-2C).
Figure 5-2: Extended leukemic latency in Prdm16-deficient MLL-AF9 cells. (A) Survival of lethally
irradiated mice transplanted with bone marrow HSC-derived MLL-AF9 cells from Vav-Cre-/- Prdm16fl/fl (WT) and Vav-Cre+/- Prdm16fl/fl (KO) mice. (B) In vitro colony-forming assays of MLL-AF9 cells corresponding to (A). (n = 4 independent assays in duplicate). (C) Survival of lethally irradiated mice transplanted with MLL-AF9 cells generated from bone marrow Lin-Sca1-kit+ cells from Vav-Cre-/- Prdm16fl/fl (WT) and
Vav-Cre+/- Prdm16fl/fl (KO) mice. (n.s = P > 0.05; * = P < 0.05; ** = P < 0.01; *** = P < 0.001, Student’s t-test for single comparisons and Gehan-Breslow-Wilcoxon test for comparison of survival curves).
We obtained similar results using MLL-AF9 transduction of HSCs from germline-deleted Prdm16-/- mice. As these mice die perinatally, fetal liver cells were used. We also assayed Prdm16+/- MLL-AF9 cells, as our previous report showed an intermediate phenotype of heterozygote HSCs indicating Prdm16 haploinsufficiency. Mice transplanted with Prdm16-/- MLL-AF9 cells had a significantly increased leukemic latency compared to WT, with heterozygotes showing an intermediate phenotype (Figure 5-3A). As with bone marrow-derived Prdm16fl/fl.Vav-Cre+ deletion, germline-deleted Prdm16-/ - MLL-AF9 cells did not, however, exhibit in vitro growth defects in colony-forming assays (Figure 5-3B). To determine if physiological levels of f-Prdm16 suppress leukemogenesis, we transduced fetal liver HSCs from
f-Prdm16-deleted Δ47-fPrdm16-/- mice with MLL-AF9. In contrast to the Prdm16-/- deletion, leukemic latency was unchanged between mice transplanted with either Δ47-fPrdm16-/- or WT cells (Figure 5-3C).
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As Δ47-fPrdm16-/- cells express normal levels of s-Prdm16, our results taken together support the conclusion that s-Prdm16 is required for normal latency of murine MLL-AF9 leukemia, whereas f-Prdm16 is not required. Furthermore, because Δ47-fPrdm16-/- MLL-AF9 cells did not have a shorter latency than WT, our data argue that physiological levels of f-Prdm16 do not have a tumor suppressor effect.
Figure 5-3: s-Prdm16 is required for efficient MLL-AF9 leukemogenesis. (A) Survival of lethally irradiated
mice transplanted with Prdm16+/+ (WT), Prdm16+/- (HET), or Prdm16-/- (KO) fetal liver HSC-derived MLL-AF9 cells. (n = 14-15 recipients, 3 independently-derived MLL-AF9 lines). (B) In vitro colony-forming assays of MLL-AF9 cells from (A) (n = 4 independent assays in duplicate). (C) Survival of mice transplanted with fetal liver HSC-derived MLL-AF9 cells from Δ47-fPrdm16-/- (KO) or WT littermate mice. (n.s = P > 0.05; * = P < 0.05; ** = P < 0.01; *** = P < 0.001, One-way ANOVA for multiple comparisons, Gehan-Breslow-Wilcoxon test for comparison of survival curves)
As MLL-AF9 leukemia from Prdm16-deficient mice exhibits a delayed latency, Prdm16 must be required in either the cell-of-origin and/or the resulting leukemic cells. We know from our previous publications that both Prdm16 isoforms are highly expressed in HSCs, the cell-of-origin in our model. We therefore performed qPCR on ex vivo MLL-AF9 cells from the bone marrow of moribund recipient mice and found that in these cells Prdm16 levels were undetectable (Figure 5-4A). Similar results were obtained with MLL-AF9 cells cultured in vitro (Figure 5-4B). Therefore, expression of s-Prdm16 in HSCs at the time of leukemic transformation is likely the determinant of MLL-AF9 latency in vivo. Previous findings in mouse models of MLL-AF9 are in accord with this conclusion, demonstrating that differences in the cell-of-origin can significantly influence latency and gene expression patterns of the resulting leukemia224. The authors
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found significant epigenetic differences in the resulting leukemic cells, leading us to speculate that
s-Prdm16 may possibly function through a similar mechanism, inducing an inheritable epigenetic
signature that promotes leukemogenesis at the time of MLL-AF9 transformation.
Figure 5-4: Undetectable expression of Prdm16 in MLL-AF9 cells. (A) Quantitative PCR showing percent
expression of Prdm16 relative to WT HSC controls in other stem and progenitor cells (MPPs and CMPs) and in ex vivo WT MLL-AF9 leukemic cells (sorted hNGFR+ cells from bone marrow of moribund mice). (n = 3, in triplicate). (B) Relative percent mRNA expression of Prdm16 in in vitro cultured MLL-AF9 cells compared to HSC controls. (n = 3, in triplicate).